Novel antibacterial and fungicidal peptide in which lysine and tryptophan residues are repeated, and use thereof

ABSTRACT

The present invention relates to an antibacterial and fungicidal peptide in which a lysine and tryptophan dipeptide is repeated. More specifically, the antibacterial and fungicidal peptide of the present invention, in which lysine and tryptophan dipeptide is repeated four times, shows excellent antibacterial activities with respect to gram-positive bacteria, gram-negative bacteria and antibiotic-resistant strains by affecting the inner membrane of harmful microorganisms, has remarkable fungicidal activities with respect to pathogenic fungi and antibiotic-resistant fungi, and shows little cytotoxicity, and thus can be useful for a pharmaceutical composition, a cosmetic composition, agricultural chemicals, a food preservative, a cosmetic preservative, and a pharmaceutical preservative.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel antibiotic peptide in whichlysine and tryptophan residues are repeated and an antibacterial andfungicidal pharmaceutical composition comprising the same as an activeingredient.

2. Description of the Related Art

The bacterial infection is one of the most common and lethal causes ofhuman disease. Unfortunately, misuse or overuse of antibiotics broughtantibiotic resistance of bacteria. In fact, the period that it takes forbacteria to show resistance against a new antibiotic is much shorterthan the period for the development of a novel antibiotic or analoguesthereof. For example, fatal bacteria Enterococcus faecalis,Mycobacterium tuberculosis and Pseudomonas aeruginosa have raised theirresistance against almost every antibiotics developed so far (Stuart B.Levy, Scientific American, 46-53, 1998).

Tolerance against antibiotics is different from resistance againstantibiotics. Antibiotic tolerance was first observed in Pneumococcus sp.in 1970s with providing an important clue to understand the mechanism ofthe action of penicillin (Tomasz et al., Nature, 227, 138-140, 1970).Such species of bacteria that show tolerance stop growing in thepresence of a common concentration of antibiotics but cannot be killed.Tolerance is developed when autolytic enzyme such as autolysin is notactivated in the presence of an antibiotic that inhibits cell wallsynthase. Penicillin kills bacteria by activating endogenous hydrolyticenzyme, but the bacteria, on the contrary, inhibit the enzyme activityto survive in the presence of antibiotics.

It is a clinically very important phenomenon that bacteria havetolerance against antibiotics. That is, once it is impossible to killthe bacteria having tolerance, the treatment effect of antibiotics oninfectious disease decreases (Handwerger and Tomasz, Rev. Infec. Dis.,7, 368-386, 1985). Tolerance is regarded as a precondition for thecoming resistance against antibiotics in bacteria. That means even afterthe treatment with antibiotics, some strains still survive. Thesestrains acquire a new genetic element to build resistance againstantibiotics, so that they can continue to survive in the presence ofantibiotics. All the strains showing resistance against antibiotics alsohave tolerance against those antibiotics as well (Liu and Tomasz, J.Infect. Dis., 152, 365-372, 1985). Therefore, it is urgent request todevelop a novel antibiotic that is able to kill the strain havingantibiotic resistance.

Tolerance is largely divided into two groups according to its mechanism.First, phenotypic tolerance is the one that is observed when the growthrate decreases in every strain (Tuomanen E., Revs. Infect. Dis., 3,S279-S291, 1986). The other one is genetic tolerance developed bymutation in some specific strains. The basic and common phenomenon inboth cases is the down regulation of autolytic enzyme activity. Thisdown regulation is temporary when it is phenotypic tolerance caused byan external stimulus, but is permanent when it is genetic tolerancecaused by mutation in regulatory pathways controlling cell hemolysis.Precisely, the most simple genetic tolerance is caused by the deletionof autolytic enzyme. It is very rare for a strain to have tolerance withthe deletion of autolytic enzyme caused by some unknown reasons.However, tolerance observed clinically in strains is mostly caused bythe phenotypic regulation of autolytic enzyme activity (Tuomanen et al.,J. infect. Dis., 158, 36-43, 1988).

As explained hereinbefore, it is required to develop a novel antibioticto eliminate antibiotic-resistant bacteria which is functioningindependently from autolytic enzyme activity.

In the meantime, bacteria can kill other neighboring bacteria bysynthesizing peptides or small organic molecules, called ‘bacteriocin’.The bacteriocin is divided into three groups according to the structuralcharacteristics; which are lantibiotics, nonlantibiotics, and thosesecreted by signal peptides (Cintas et al., J. Bad., 180, 1988-1994,1998). Animals including insects can self-produce such peptideantibiotics (Bevins et al., Ann. Rev. Biochem., 59, 395-414, 1990),which are also divided into three groups according to the structure.First group is cysteine-rich β-sheet peptide, the second group isα-helical amphiphilic peptide, and the third group is proline-richpeptide (Mayasaki et al., Int. J. Antimicrob. Agents, 9, 269-280, 1998).Such antibacterial peptides are known to play an important role in hostdefense and innate immune system (Boman, H. G., Cell, 65:205, 1991;Boman, H. G., Annu. Rev. Microbiol., 13:61, 1995). The saidantibacterial peptides have different structures determined by the aminoacid sequences, among which the most common structure is α-helicalamphiphilic form without cysteine residues, as shown in cecropin, theantibacterial peptide found in insects.

The antibacterial activity of amphiphilic peptide has been well studied,based on which the attempts have been made to develop antibiotics.Melittin is the peptide taking at least 50% of solid components of beevenom, in which C-terminal is amidated and cytotoxicity againsteukaryotic cell is strong enough to destroy mammalian cells even at alow concentration and antibacterial activity against such microorganismsas Gram-negative bacteria and Gram-positive bacteria is excellent(Habermann, E., Science, 177: 314, 1972; Steiner, H., et al., Nature,292: 246, 1981; Tosteson, M. T., et al., Biochemistry, 228: 337, 1987).Cecropin amphiphilic peptide having HP(2-20) like amino acid sequencewas first found in fruit fly and found later on in silkworm pupa and insmall intestine of pig. Particularly, according to the previous reports,cecropin A (CA) exhibits high antibacterial activity but has only minorantifungal and anticancer activity (Boman, H. G. and Hultmark, D., Annu.Rev. Microbiol., 41: 103, 1987).

In addition to the studies on the activity of the mentioned amphiphilicpeptide, the amino acid sequence and protein structure have also beenstudied, saying that the characteristics of the sequence are closelyrelated to the antibacterial activity. For example, when arginine issubstituted with lysine therein, the activity still remains butcytotoxicity against eukaryotic cell is reduced (Epand R F, Lehrer R I,Waring A, Wang W, Maget-Dana R, Lelievre D, Epand R M., Bioploymers2003; 71: 2-16.; Park Y, Lee D G, Jang S H, Woo E R, Jeong H G, Choi CH, Hahm K S., Biochim. Biophys. Acta 2003; 1645: 172-182.). A strongsynthetic antibacterial peptide composed of lysine residues alone islocated on the polar surface of amphiphilic structure and interacts withthe upper section of anionic lipid, which gives the specificity tomicroorganism (Dathe M, Schumann M, Wieprecht T, Winkler A, Beyermann M,Krause E, Matsuzaki K, Murase O, Bienert M., Biochemistry 1996; 35:12612-12622.; Song Y M, Park Y, Lim S S, Yang S T, Woo E R, Park I S,Lee J S, Kim J I, Hahm K S, Kim Y, Shin S Y., Biochemistry 2005; 44:12094-12106.; Gopal R, Park S C, Ha K J, Cho S J, Kim S W, Song P I, NahJ W, Park Y, Hahm K S., J. Pept. Sci. 2009; 15: 589-594.).

Therefore, the present inventors synthesized peptides in variouslengths, in which lysine and tryptophan residues form KW dipeptides thatare repeated 2-5 times. The present inventors eventually completed thisinvention by confirming that the peptides of the invention, particularly(KW)₄ represented by SEQ. ID. NO: 3 had excellent antibacterial activityagainst Gram-positive, Gram-negative, and antibiotic-tolerant strainsand excellent fungicidal activity against pathogenic fungi andantibiotic-tolerant fungi by affecting the inner membrane of thepathogenic microorganism but has least cytotoxicity, suggesting that thepeptides of the present invention can be functioning as safeantibacterial and fungicidal drugs to humans.

The present inventors also investigated the antibacterial activity of(KW)₃ in which lysine and tryptophan residues are repeated three times(Gopal R, Kim Y J, Seo C H, Hahm K S, Park Y., J Pept Sci. 2011; 17:329-334). As a result, it was confirmed that (KW)₄ of the inventiondemonstrated 16 times higher antibacterial activity in general strainsand times higher antibacterial activity in antibiotic-tolerant strainsthan (KW)₃, along with excellent antifungal activity. In conclusion,(KW)₄ was confirmed to be a better peptide in its antibacterial andantifungal activity than (KW)₃.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an antibacterial andfungicidal peptide in which lysine and tryptophan residues are repeated.

It is another object of the present invention to provide anantibacterial and fungicidal composition comprising the saidantibacterial and fungicidal peptide.

To achieve the above objects, the present invention provides anantibacterial or fungicidal peptide in which lysine and tryptophan arerepeated.

The present invention also provides an antibacterial or fungicidalpharmaceutical composition comprising the said peptide as an activeingredient.

The present invention also provides an antibacterial or fungicidalcosmetic composition comprising the said peptide as an activeingredient.

The present invention also provides a non-toxic agricultural pesticidecomprising the said peptide as an active ingredient.

The present invention also provides a preservative for medicine,cosmetics, food, or feed comprising the said peptide as an activeingredient.

The present invention also provides a method for suppressing bacteria orfungi containing the step of treating a pharmaceutically effective doseof the said antibacterial or fungicidal pharmaceutical composition tothe target bacteria or fungi to suppress them.

The present invention also provides a method for treating the pathogenicbacteria or fungi caused disease containing the step of administering apharmaceutically effective dose of the said antibacterial or fungicidalpharmaceutical composition to a subject having the pathogenic bacteriaor fungi caused disease.

The present invention also provides a method for preventing thepathogenic bacteria or fungi caused disease containing the step ofadministering a pharmaceutically effective dose of the saidantibacterial or fungicidal pharmaceutical composition to a subject.

The present invention also provides a use of the said peptide for thepreparation of an antibacterial or fungicidal pharmaceuticalcomposition.

The present invention also provides a use of the said peptide for thepreparation of an antibacterial or fungicidal cosmetic composition.

The present invention also provides a use of the said peptide for thepreparation of a non-toxic agricultural pesticide.

The present invention also provides a use of the said peptide for thepreparation of a preservative for medicine, cosmetics, food, or feed.

The present invention also provides the peptide usable for thesuppression of pathogenic bacteria or fungi.

In addition, the present invention provides the peptide usable fortreating or preventing the pathogenic bacteria or fungi caused disease.

Advantageous Effect

As explained hereinbefore, the antibacterial and fungicidal peptide ofthe present invention in which lysine and tryptophan residues arerepeated demonstrates excellent antibacterial activity againstGram-positive, Gram-negative, and antibiotic-tolerant strains andexcellent fungicidal activity against pathogenic fungi andantibiotic-tolerant fungi by affecting the inner membrane of thepathogenic microorganism but has least cytotoxicity, so that it can beeffectively used as a pharmaceutical composition, a cosmeticcomposition, an agricultural pesticide, a food preservative, a cosmeticpreservative, and a medicine preservative.

BRIEF DESCRIPTION OF THE DRAWINGS

The application of the preferred embodiments of the present invention isbest understood with reference to the accompanying drawings, wherein:

FIG. 1 is a diagram illustrating the peptide of the present inventionand the sequence thereof.

FIGS. 2A and 2B are graphs illustrating the maximum fluorescent lightemission wavelength of tryptophan residue in the peptide of theinvention measured in order to analyze aggregation over theconcentration of the peptide in water and PBS. FIGS. 2C and 2D aregraphs illustrating the secondary structure of the peptide in PBSanalyzed by circular dichroism (CD).

A: maximum fluorescent light emission wavelength values of (KW)₄ and(KW)₅ in water

B: maximum fluorescent light emission wavelength values of (KW)₄ and(KW)₅ in PBS

* (KW)₄(), (KW)₅(▴)

C: (KW)₄ structure in PBS

D: (KW)₅ structure in PBS

* 25 μM(), 50 μM(▪), 100 μM(▴), 150 μM(♦)

FIG. 3 is a diagram illustrating the acting location of the peptide ofthe present invention, for which the peptide was labeled with afluorescent material and then treated to bacteria.

A: antibiotic-tolerant E. coli (E. coli CCARM 1229)

B: antibiotic-tolerant Staphylococcus aureus (S. aureus CCARM 3090)

FIG. 4 is a graph illustrating the effect of the peptide of the presentinvention on bacterial membrane and the duration of bactericidal actionaccording to the concentration of the peptide.

A membrane depolarization of antibiotic-tolerant E. coli (E. coli CCARM1229)

B: membrane depolarization of antibiotic-tolerant Staphylococcus aureus(S. aureus CCARM 3090)

C: effect on inner membrane of E. coli

D: effect on inner membrane of Staphylococcus aureus (S. aureus)

E: antibacterial activity against antibiotic-tolerant E. coli (E. coliCCARM 1229)

F: antibacterial activity against antibiotic-tolerant Staphylococcusaureus (S. aureus CCARM 3090)

* A˜D: (KW)₂(♦), (KW)₃(▪), (KW)₄(), (KW)₅(▴), Melittin(∘)

E˜F: (KW)₄ MIC(□), (KW)₄ 2MIC(Δ), (KW)₅ MIC(▪), (KW)₅ 2MIC(▴)

FIG. 5 is a graph illustrating the emission of the fluorescent materialand the cohesiveness measured in order to confirm whether or not thepeptide of the present invention could work on the bacteria-like doublemembrane.

A: PE:PG (7:3, w/w)

B: PC:CH (10:1, w/w)

C: PE:PG (7:3, w/w)

* (KW)₂(♦), (KW)₃(▪), (KW)₄(), (KW)₅(▴)

FIG. 6 is a graph illustrating the secondary structure of the peptide ofthe present invention in the presence of the bacteria-like doublemembrane analyzed by circular dichroism (CD).

A: PE:PG (7:3, w/w)

B: PC:CH (10:1, w/w)

* (KW)₂(♦), (KW)₃(▪), (KW)₄(), (KW)₅(▴)

FIG. 7 is an electron micrograph illustrating the activity of thepeptide of the present invention on antibiotic-tolerant E. coli (E. coliCCARM 1229) treated with the peptide.

A: negative control (non-treated)

B: (KW)₄

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, the present invention is described in detail.

The present invention provides an antibacterial and fungicidal peptidein which the lysine and tryptophan (KW) dipeptide is repeated.

The lysine and tryptophan KW dipeptide is preferably repeated 1-10times, and more preferably 4 times, or at least 4 times, but not alwayslimited thereto. In particular embodiments the KW dipeptide is repeated4-10 times, or 4-5 times.

The said peptide is composed of the amino acid sequence represented bySEQ. ID. NO: 3; hardly has cytotoxicity; is easy to be synthesizedbecause of its short length comprising the minimum 8-mer amino acids;and has amphiphilicity owing to the enlarged hydrophobic region, but notalways limited thereto.

The said peptide has the antibacterial activity preferably againstGram-positive bacteria, Gram-negative bacteria, and antibiotic-tolerantstrains, and more precisely the Gram-positive bacteria are selected fromthe group consisting of Bacillus subtilis, Staphylococcus aureus andListeria monocytogenes, and the Gram-negative bacteria are selected fromthe group consisting of Escherichia coli, Psedomonas aeruginosa andSalmonella typhimurium. In the meantime, the antibiotic-tolerant strainsare preferably selected from the group consisting of E. coli (E. coliCCARM 1229, 1238), Salmonella (S. typhimurium CCARM 8007, 8009, 8013)and Staphylococcus (S. aureus CCARM 3089, 3090, 3108, 3114, 3126), butnot always limited thereto.

The said peptide has the fungicidal activity preferably againstpathogenic fungi and antibiotic-tolerant fungi, and more precisely thesaid pathogenic fungi are selected from the group consisting of Candidaalbicans, C. catenulate, C. intermidia, C. rugosa, C. glabrata and C.melibiosica, and the antibiotic-tolerant fungi are preferably C.albicans CCARM 14001, 14007, and 14020, but not always limited thereto.

In addition, the said peptide is conjugated to the outer wall ofantibiotic-tolerant E. coli (E. coli CCARM 1229) and antibiotic-tolerantStaphylococcus aureus (S. aureus CCARM 3090) in order to be functioningthere and further invades into the inner wall to affect them. Thus, whenthe peptide is treated to antibiotic-tolerant E. coli (E. coli CCARM1229), it makes the surface of the strain rough and injured to causeaggregation there, but the function is not always limited thereto.

To prepare a novel antibacterial and fungicidal peptide, the presentinventors synthesized the peptide which is short and has a simplesequence composed of only two kinds of amino acids, lysine andtryptophan. As a result, the inventors synthesized (KW)₂ (SEQ. ID.NO: 1) in which lysine and tryptophan residues are repeated twice, (KW)₃(SEQ. ID. NO: 2) in which those residues are repeated three times, (KW)₄(SEQ. ID. NO: 3) in which those residues are repeated four times, and(KW)₅ (SEQ. ID. NO: 4) in which those residues are repeated 5 times (seeFIG. 1).

To investigate the antibacterial activity of the peptide of the presentinvention, the present inventors used (KW)_(n) (n=2, 3, or 5) peptidesin different lengths and Melittin peptide as controls. Minimalinhibitory concentration (referred as “MIC” hereinafter) that was theconcentration each strain could survive without being divided wasmeasured from all the groups. At this time, Gram-positive bacteria usedfor the investigation were Bacillus subtilis, Staphylococcus aureus andListeria monocytogenes, and Gram-negative bacteria used for themeasurement were Escherichia coli, Psedomonas aeruginosa and Salmonellatyphimurium. Also, the antibiotic-tolerant strains used herein were E.coli (E. coli CCARM 1229, 1238), Salmonella (S. typhimurium CCARM 8007,8009, 8013) and Staphylococcus (S. aureus CCARM 3089, 3090, 3108, 3114,3126). As a result, the peptide of the present invention represented bySEQ. ID. NO: 3 demonstrated antibacterial activity in the generalstrains 64 times higher than the control peptide represented by SEQ. ID.NO: 1, 16 times higher than the peptide represented by SEQ. ID. NO: 2,and 8 times higher than the peptide represented by SEQ. ID. NO: 4. Inthe meantime in the antibiotic-tolerant strains, the peptide of thepresent invention showed the antibacterial activity 32 times, times, and8 times higher than the above peptides, respectively (see Table 1).Therefore, the peptide of the present invention was confirmed to haveexcellent antibacterial activity against all the Gram-positive bacteria,Gram-negative bacteria, and antibiotic-tolerant strains.

To investigate the fungicidal activity of the peptide of the presentinvention, MIC was measured by using pathogenic fungi such as Candidaalbicans, C. catenulate, C. intermidia, C. rugosa, C. glabrata and C.melibiosica and antibiotic-tolerant fungi such as C. albicans CCARM14001, 14007 and 14020. As a result, the peptide of the presentinvention represented by SEQ. ID. NO: 3 demonstrated excellentfungicidal activity regardless of solution used for the experiment andthe peptide represented by SEQ. ID. NO: 4 showed excellent fungicidalactivity in PBS (see Table 2). The peptides each represented by SEQ. ID.NO: 3 and NO: 4 were confirmed to have excellent fungicidal activityagainst antibiotic-tolerant fungi (see Table 3).

The present inventors also investigated cytotoxicity of the peptide ofthe invention. To do so, hemolytic activity of the peptide was measured.As a result, the peptide represented by SEQ. ID. NO: 3 hardly showedcytotoxicity (see Table 4).

The present inventors also investigated aggregation and the structure ofthe peptide by using a spectrofluorometer and circular dichroism (CD).As a result, the peptide represented by SEQ. ID. NO: 3 did not showaggregation in water nor in PBS, but the peptide represented by SEQ. ID.NO: 4 showed aggregation in PBS (see FIG. 2).

To investigate the acting site of the peptide of the present inventionin E. coli, confocal laser scanning microscopy was used. As a result, itwas observed that the peptide represented by SEQ. ID. NO: 3 wasconjugated to the outer wall of antibiotic-tolerant E. coli (E. coliCCARM 1229) and antibiotic-tolerant Staphylococcus aureus (S. aureusCCARM 3090) (see FIG. 3).

The present inventors also investigated whether or not the peptide ofthe invention could actually affect the membrane of E. coli and how fastthe peptide could kill the bacteria by performing depolarizationanalysis, observation of action on inner cell membrane, and measurementof apoptosis over the time. As a result, the peptide represented by SEQ.ID. NO: 1 could not induce depolarization in antibiotic-tolerant E. coli(E. coli CCARM 1229) nor in antibiotic-tolerant Staphylococcus aureus(S. aureus CCARM 3090). The peptide represented by SEQ. ID. NO: 2induced depolarization approximately 20% at the concentration of 50 μM,while the peptides represented by SEQ. ID. NO: 3 and SEQ. ID. NO: 4induced depolarization approximately 80% at the concentration of 25 μMin both strains (see A and B of FIG. 4). The peptides represented bySEQ. ID. NO: 3 and SEQ. ID. NO: 4 were able to affect inner membrane ofE. coli and Staphylococcus aureus (see C and D of FIG. 4). The peptiderepresented by SEQ. ID. NO: 3 showed faster antibacterial activityagainst antibiotic-tolerant E. coli (E. coli CCARM 1229) andantibiotic-tolerant Staphylococcus aureus (S. aureus CCARM 3090) thanthe peptide represented by SEQ. ID. NO: 4 did.

The present inventors also investigated whether or not the peptide ofthe invention could take direct action on bacteria and erythrocyte-likeliposome and whether or not the peptide could coagulate bacteria-likeliposome by using a spectrofluorometer. As a result, both the peptiderepresented by SEQ. ID. NO: 3 and the peptide represented by SEQ. ID.NO: 4 took action against bacteria-like liposome in the presence ofL-a-phosphatidylethanolamine (PE) and L-a-phosphatidylglycerol (PG)(7:3). However, in the presence of egg yolk L-a-phosphatidylcholine (PC)and cholesterol (CH) at the ratio of 10:1, the peptide represented bySEQ. ID. NO: 3 emitted the fluorescence less than half the amountemitted by the peptide represented by SEQ. ID. NO: 4 at the sameconcentration (see A and B of FIG. 5). The peptide represented by SEQ.ID. NO: 4 induced coagulation of liposome more than the peptiderepresented by SEQ. ID. NO: 3 (see FIG. 5C).

The present inventors also investigated how the structure of the peptideof the invention could be changed in the course of the interaction withbacteria and erythrocyte-like liposome by using circular dichroism (CD).As a result, the peptides each represented by SEQ. ID. NO: 1 and SEQ.ID. NO: 2 did not form any specific structure in bacteria nor inerythrocyte-like liposome, while the peptides represented by SEQ. ID.NO: 3 and SEQ. ID. NO: 4 showed structural change with folding (see FIG.6). This phenomenon is regarded as an important factor to breakbacterial membrane.

Further, the present inventors investigated the mechanism ofantibacterial activity of the peptide of the invention inantibiotic-tolerant E. coli (E. coli CCARM 1229) by using scanningelectron microscope. As a result, when E. coli was treated with thepeptide represented by SEQ. ID. NO: 3, the smooth surface of theantibiotic-tolerant E. coli turned into rough and injured with showingaggregation as well (see FIG. 7).

Therefore, the peptide of the present invention in which lysine andtryptophan residues are repeated 4 times demonstrates excellentantibacterial activity and fungicidal activity by affecting the innermembrane of the pathogenic microorganism but has least cytotoxicity,suggesting that the peptide of the invention can be effectively used asa safe antibacterial agent for human.

The present invention also provides an antibacterial or fungicidalpharmaceutical composition comprising the peptide of the invention as anactive ingredient.

The present invention also provides a use of the said peptide for thepreparation of an antibacterial or fungicidal pharmaceuticalcomposition.

The present invention also provides the peptide usable for thesuppression of pathogenic bacteria or fungi.

The present invention also provides a method for suppressing bacteria orfungi containing the step of treating a pharmaceutically effective doseof the said antibacterial or fungicidal pharmaceutical composition tothe target bacteria or fungi to suppress them.

The said pharmaceutical composition has the antibacterial activitypreferably against Gram-positive bacteria, Gram-negative bacteria, andantibiotic-tolerant strains, and more precisely the Gram-positivebacteria are selected from the group consisting of Bacillus subtilis,Staphylococcus aureus and Listeria monocytogenes, and the Gram-negativebacteria are selected from the group consisting of Escherichia coli,Psedomonas aeruginosa and Salmonella typhimurium. In the meantime, theantibiotic-tolerant strains are preferably selected from the groupconsisting of E. coli (E. coli CCARM 1229, 1238), Salmonella (S.typhimurium CCARM 8007, 8009, 8013) and Staphylococcus (S. aureus CCARM3089, 3090, 3108, 3114, 3126), but not always limited thereto.

The said pharmaceutical composition has the fungicidal activitypreferably against pathogenic fungi and antibiotic-tolerant fungi, andmore precisely the said pathogenic fungi are selected from the groupconsisting of Candida albicans, C. catenulate, C. intermidia, C. rugosa,C. glabrata and C. melibiosica, and the antibiotic-tolerant fungi arepreferably C. albicans CCARM 14001, 14007, and 14020, but not alwayslimited thereto.

The peptide of the present invention in which lysine and tryptophanresidues are repeated demonstrates excellent antibacterial activity andfungicidal activity by affecting the inner membrane of the pathogenicmicroorganism but has least cytotoxicity, suggesting that the peptide ofthe invention can be effectively used as an antibacterial and fungicidalpharmaceutical composition.

The novel peptide of the present invention can be administered orally orparenterally and be used in general forms of pharmaceutical formulation.

That is, the novel peptide of the present invention can be prepared fororal or parenteral administration by mixing with generally used diluentsor excipients such as fillers, extenders, binders, wetting agents,disintegrating agents and surfactant. Formulations for parenteraladministration are sterilized aqueous solutions, water-insolubleexcipients, suspensions, emulsions, lyophilized preparations,suppositories and injections. Water insoluble excipients and suspensionscan contain, in addition to the active compound or compounds, propyleneglycol, polyethylene glycol, vegetable oil like olive oil, injectableester like ethylolate, etc. Suppositories can contain, in addition tothe active compound or compounds, witepsol, macrogol, tween 61, cacaobutter, laurin butter, glycerogelatin, etc.

The novel peptide of the present invention can be mixed with manypharmaceutically acceptable carriers such as physiological saline ororganic solvent, and can additionally include carbohydrates such asglucose, sucrose or dextran, antioxidants such as ascorbic acid orglutathion, chelating agents, low molecular proteins or otherstabilizers to enhance stability or absorptiveness.

The effective dose of the novel peptide of the present invention is0.01˜100 mg/kg, and more preferably 0.14˜1.4 mg/kg, which can beadministered 1˜3 times a day.

The present invention also provides a method for treating the pathogenicbacteria or fungi caused disease containing the step of administering apharmaceutically effective dose of the said antibacterial or fungicidalpharmaceutical composition to a subject having the pathogenic bacteriaor fungi caused disease.

The present invention also provides a method for preventing thepathogenic bacteria or fungi caused disease containing the step ofadministering a pharmaceutically effective dose of the saidantibacterial or fungicidal pharmaceutical composition to a subject.

The present invention also provides the peptide usable for treating orpreventing the pathogenic bacteria or fungi caused disease.

The effective dose of the novel peptide of the present invention can beadministered in the form of bolus, by single dose having relativelyshort period of infusion or by multiple dose of fractionated treatmentprotocol for a long term. The decision of the effective dose depends onthe administration pathway, treatment times, age and other conditions ofa patient, etc. Therefore, any expert who has knowledge on this fieldcan decide the effective dose of the novel peptide of the presentinvention.

The present invention also provides an antibacterial or fungicidalcosmetic composition comprising the peptide of the invention as anactive ingredient.

The present invention also provides a use of the said peptide for thepreparation of an antibacterial or fungicidal cosmetic composition.

The peptide of the present invention in which lysine and tryptophanresidues are repeated demonstrates excellent antibacterial activity andfungicidal activity by affecting the inner membrane of the pathogenicmicroorganism but has least cytotoxicity, suggesting that the peptide ofthe invention can be effectively used as an antibacterial and fungicidalcosmetic composition.

The cosmetic composition of the present invention can include, inaddition to the said novel peptide, any conventional ingredientsgenerally used in cosmetics, for example such additives and carriers asstabilizers, solubilizers, vitamins, pigments and flavors, but notalways limited thereto.

The cosmetic composition of the present invention can be formulated inany form that can be accepted in the art, which is exemplified bysolution, suspension, emulsion, paste, gel, cream, lotion, powder, soap,surfactant-containing cleansing, oil, powdered foundation, emulsifiedfoundation, wax foundation and spray, but not always limited thereto.Particularly, the cosmetic composition of the present invention can beprepared in the form of soft lotion (skin), nutrition lotion (milklotion), nutrition cream, massage cream, essence, eye cream, cleansingcream, cleansing foam, cleansing water, pack, spray or powder.

In the case that the cosmetic composition is formulated as paste, creamor gel, the proper carrier can be selected from the group consisting ofanimal oil, vegetable oil, paraffin, starch, tracanth, cellulosederivative, polyethylene glycol, silicon, bentonite, silica, talk andzinc oxide.

In the case that the cosmetic composition is formulated as powder orspray, the proper carrier can be selected from the group consisting oflactose, talc, silica, aluminum hydroxide, calcium silicate andpolyamide powder, and in particular if the composition of the presentinvention is formulated as spray, a propellant such aschlorofluorohydrocarbon, propane/butane or dimethyl ether can beadditionally included.

In the case that the cosmetic composition is formulated as liquid oremulsion, the proper carrier can be selected from the group consistingof solvent, solubilizer and emulsifier, which is exemplified by water,ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerolaliphatic ester, polyethylene glycol and fatty acid ester of sorbitan.

In the case that the cosmetic composition is formulated as suspension,the proper carrier can be selected from the group consisting of liquiddiluent such as water, ethanol or propylene glycol; suspending agentsuch as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esterand polyoxyethylene sorbitan ester; microcrystalline cellulose; aluminummethahydroxide; bentonite; agar; and tragacanth.

In the case that the cosmetic composition is formulated assurfactant-containing cleansing, the proper carrier can be selected fromthe group consisting of aliphatic alcohol sulfate, aliphatic alcoholether sulfate, sulfosuccinic monoester, isethionate, imidazolinumderivative, methyltaurate, sarcosinate, fatty acid amide ether sulfate,alkyl amidobetain, aliphatic alcohol, fatty acid glyceride, fatty aciddiethanolamide, vegetable oil, lanolin derivative and ethoxylatedglycerol fatty acid ester.

The present invention also provides a non-toxic agricultural pesticidecomprising the peptide of the invention as an active ingredient.

The present invention also provides a use of the said peptide for thepreparation of a non-toxic agricultural pesticide.

The peptide of the present invention in which lysine and tryptophanresidues are repeated demonstrates excellent antibacterial activity andfungicidal activity by affecting the inner membrane of the pathogenicmicroorganism but has least cytotoxicity, suggesting that the peptide ofthe invention can be effectively used as a non-toxic agriculturalpesticide.

The present invention also provides a preservative for medicine,cosmetics, food, or feed comprising the peptide of the invention as anactive ingredient.

The present invention also provides a use of the said peptide for thepreparation of a preservative for medicine, cosmetics, food, or feed.

The peptide of the present invention in which lysine and tryptophanresidues are repeated demonstrates excellent antibacterial activity andfungicidal activity by affecting the inner membrane of the pathogenicmicroorganism but has least cytotoxicity, suggesting that the peptide ofthe invention can be effectively used as a preservative for medicine,cosmetics, food, or feed.

The preservative for food, cosmetics, or medicine is the additive usedto prevent spoilage, decomposition, and chemical changes, which includesgermicides and antioxidants, etc. Functional antibiotics suppressing thegrowth of microorganisms including bacteria, fungi, and yeasts can bealso included to prevent the growth of spoilage microorganisms inmedicine and food. Such preservative for food, cosmetics, or medicine isexpected to be non-toxic and is effective even at a low concentration.Agricultural pesticides to prevent damages by blight and insects inagricultural crops are also expected to inhibit the growth of harmfulmicroorganisms but expected to be non-toxic to human in order for humanto eat the agricultural product safely.

Practical and presently preferred embodiments of the present inventionare illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

Example 1 Peptide Synthesis, Separation and Purification

To synthesize the peptide having the repeats of lysine and tryptophan,the present inventors used Merrifield's liquid-solid method (Merrifield,R B., J. Am. Chem. Soc., 85, 2149, 1963) using Fmoc (9-fluorenylmethoxycarbonyl) as the amino acid protecting group. Particularly, Rink AmideMBHA-Resin was used as a starting material for the peptide having —NH₂type C-terminal, and Fmoc-amino acid-Wang Resin (Wang Resin) was used asa starting material for the peptide having —OH type C-terminal. Thepeptide chain extension via Fmoc-amino acid coupling was performed byDCC (N-hydroxybenzo triazole(HOBt)-dicyclo-hexycarbodiimide) method.After Fmoc-amino acids at N-terminal of each peptide were coupled, Fmocgroup was eliminated by using NMP (20% piperidine/N-methylpyrolidone)solution, followed by washing with NMP and DCM (dichoromethane) severaltimes and drying over nitrogen gas. TFA (trifluoroaceticacid)-phenol-thioanisole-H₂O-triisopropylsilane (85:5:5:2.5:2.5,vol./vol.) solution was added thereto, followed by reaction for 2˜3hours to eliminate the protecting group and separate the peptide fromthe regin. The peptide was precipitated by using diethylether. The crudepeptide obtained thereby was purified by reverse phase (RP)—HPLC column(Delta Pak, C₁₈ 300 Å, 15, 19.0 mm×30 cm, Waters) in acetonitrilegradient containing 0.05% TFA. The synthesized peptide was hydrolyzedwith 6N HCl at 110° C., and the residue was concentrated under reducedpressure. After dissolving the concentrate in 0.02 N HCl, amino acidcomposition was analyzed with an amino acid analyzer (Hitachi 8500 A).

As a result, as shown in FIG. 1, the peptide having the wanted aminoacid sequence was successfully synthesized (FIG. 1).

Example 2 Measurement of Antibacterial Activity of Peptide <2-1>Measurement of MIC in Bacteria

To measure the antibacterial activity of the peptide prepared in Example1, the present inventors measured MIC, the minimal concentration thatcan keep the strain without being divided. For the measurement ofantibacterial activity, Gram-positive bacteria selected from the groupconsisting of Bacillus subtilis, Staphylococcus aureus and Listeriamonocytogenes, Gram-negative bacteria selected from the group consistingof Escherichia coli, Psedomonas aeruginosa and Salmonella typhimurium,and antibiotic-tolerant strains selected from the group consisting of E.coli (E. coli CCARM 1229, 1238), Salmonella (S. typhimurium CCARM 8007,8009, 8013) and Staphylococcus (S. aureus CCARM 3089, 3090, 3108, 3114,3126) were used. After cultured in optimal media, the strains werediluted at the density of 2×10⁵ cells/ml, which were then inoculated onmicrotitrate plates. The peptide synthesized in Example 1 and thepositive control peptide (Melittin) were 2-fold diluted in sodiumphosphate buffer and phosphate buffered saline (PBS) from 200 μM/well,which were added in the plate, followed by culture at 37° C. for 18˜24hours. OD₆₂₀ was measured with a microtitrate plate reader to determineMIC of each strain. The results are presented in Table 1 (the number inbracket indicates the one obtained from PBS).

As a result, as shown in Table 1, the peptide represented by SEQ. ID.NO: 3 of the present invention displayed 64 times higher antibacterialactivity in general strains than the peptide represented by SEQ. ID. NO:1, 16 times higher antibacterial activity than the peptide representedby SEQ. ID. NO: 2, and 8 times higher antibacterial activity than thepeptide represented by SEQ. ID. NO: 4. In the meantime, inantibiotic-tolerant strains, the peptide of the present inventiondemonstrated 32 times higher antibacterial activity than the peptiderepresented by SEQ. ID. NO: 1, 8 times higher antibacterial activitythan the peptide represented by SEQ. ID. NO: 2, and 8 times higherantibacterial activity than the peptide represented by SEQ. ID. NO:(Table 1).

TABLE 1 MIC of peptide in bacteria MIC (μM) SEQ. ID. SEQ. ID. SEQ. ID.SEQ. ID. Microorganism NO: 1 NO: 2 NO: 3 NO: 4 Melittin AmpicillinOxacillin Gram negative bacteria E. coli >200  50 6.25 50 1.56 — —(>200) (200) (12.5) (12.5) (1.56) S. typhimurium  50    3.12 1.56 6.250.39 — —  (200)    (6.25) (1.56) (3.12) (0.39) P. aeruginosa >200   12.53.12 25 3.12 — — (>200)  (25) (3.12) (6.25) (3.12) Gram positivebacteria S. aureus >200  50 6.25 25 1.56 — — (>200) (200) (12.5) (12.5)(1.56) B. subtilis >200 100 6.25 12.5 1.56 — — (>200) (200) (12.5)(12.5) (3.12) L. monocytogenes   200   12.5 3.12 3.12 1.56 — — (>200) (25) (3.12) (3.12) (1.56) antibiotic-tolerant strains E. coli >200  256.25 50 3.12 >200 CCARM 1229^(a) (>200) (100) (12.5) (12.5) (3.12)(>200) E. coli >200  50 12.5 50 1.56 >200 CCARM 1238^(a) (>200) (200)(12.5) (12.5) (1.56) (>200) S. typhimurium >200  50 12.5 25 6.25 >200CCARM 8007^(b) (>200) (200) (12.5) (12.5) (12.5) (>200) S.typhimurium >200  50 6.25 25 6.25 >200 CCARM 8009^(b) (>200) (200)(12.5) (12.5) (12.5) (>200) S. typhimurium >200  50 6.25 25 3.12 >200CCARM 8013^(b) (>200) (200) (12.5) (12.5) (6.25) (>200) S. aureus >200 50 12.5 50 1.56 — >200 CCARM 3089^(c) (>200) (200) (12.5) (12.5) (1.56)(>200) S. aureus >200  50 12.5 25 3.12 — >200 CCARM 3090^(c) (>200)(200) (12.5) (12.5) (3.12) (>200) S. aureus >200  50 12.5 50 1.56 — >200CCARM 3108^(c) (>200) (200) (12.5) (12.5) (1.56) (>200) S. aureus >200 50 12.5 25 3.12 — >200 CCARM 3114^(c) (>200) (200) (12.5) (12.5) (3.12)(>200) S. aureus >200  50 12.5 25 1.56 — >200 CCARM 3126^(c) (>200)(200) (12.5) (12.5) (1.56) (>200)

<2-2> Measurement of MIC in Fungi

To measure the fungicidal activity of the peptide prepared in Example 1,the present inventors measured MIC, the minimal concentration that cankeep the strain without being divided. For the measurement of fungicidalactivity, pathogenic fungi selected from the group consisting of Candidaalbicans, C. catenulate, C. intermidia, C. rugosa, C. glabrata and C.melibiosica were used. After cultured in optimal media, the strains werediluted at the density of 2×10⁵ cells/ml, which were then inoculated onmicrotitrate plates. The peptide synthesized in Example 1 and thepositive control peptide (Melittin) were 2-fold diluted in solution 1(10 mM sodium phosphate buffer, pH 7.2) and solution 2 (phosphatebuffered saline) from 128 μM/well, which were added in the plate,followed by culture at 37° C. for 18˜24 hours. OD₆₂₀ was measured with amicrotitrate plate reader to determine MIC of each strain. The resultsare presented in Table 2.

As a result, as shown in Table 2, the peptide of the present inventionrepresented by SEQ. ID. NO: 3 demonstrated excellent fungicidal activityin any solution, while the peptide represented by SEQ. ID. NO: 4 showedbetter fungicidal activity in solution 2 (phosphate buffered saline)(Table 2).

TABLE 2 MIC of peptide in fungi MIC (μM) SEQ. ID. SEQ. ID. SEQ. ID. SEQ.ID. NO: 1 NO: 2 NO: 3 NO: 4 Melittin Microorganism Sol. 1 Sol. 2 Sol. 1Sol. 2 Sol. 1 Sol. 2 Sol. 1 Sol. 2 Sol. 1 Sol. 2 C. albicans >128 >128 832 8 8 16 8 4 4 C. catenulate >128 >128 8 16 8 8 32 8 4 8 C.intermidia >128 >128 8 32 8 8 32 4 4 4 C. rugosa >128 >128 8 32 8 8 32 84 8 C. glabrata >128 >128 16  32 8 8 16 4 4 4 C. melibiosica >128 >128 832 8 8 32 8 4 8

To measure the fungicidal activity of the peptide of the presentinvention in antibiotic-tolerant fungi, MIC of the peptide was measuredby the same manner as described in Example <2-2> using RMPI 1640 andantibiotic-tolerant Candida albicans (C. albicans CCARM 14001, 14007,14020). The results are presented in Table 3.

As a result, as shown in Table 3, the peptides represented by SEQ. ID.NO: 3 and SEQ. ID. NO: 4 demonstrated excellent fungicidal activity(Table 3).

TABLE 3 MIC of peptide in antibiotic-tolerant fungi MIC (μM) SEQ. ID.SEQ. ID. SEQ. ID. SEQ. ID. Microorganism NO: 1 NO: 2 NO: 3 NO: 4Melittin Fluconazole C. albicans >128 >128 32 8 16  >128 CCARM 14001 C.albicans >128 >128 32 16  8 >128 CCARM 14007 C. albicans >128 >128 32 88 >128 CCARM 14020

Example 3 Measurement of Cytotoxicity of Peptide

To investigate whether or not the peptide of the present invention hadcytotoxicity, the present inventors investigated hemolytic activity ofthe peptide. Particularly, human erythrocytes were diluted in phosphatebuffered saline (PBS, pH 7.0) at the concentration of 8%, to which thepeptide 2-fold diluted from 200 μM/well was added, followed by reactionat 37° C. for 1 hour. Centrifugation was performed at 1,000 g and thelevel of hemoglobin included in the supernatant was measured bymeasuring OD₄₁₄. To examine hemolysis, 1% triton X-100 was added tohuman erythrocytes, followed by measuring absorbance of the supernatant.The hemolytic activity of 1% triton X-100 was regarded as 100%, withwhich hemolytic activity of each antibacterial peptide was compared. Theresult was presented by the numbers calculated by the followingmathematical formula 1.

hemolysis=(absorbance A absorbance B/absorbance A−absorbanceB)×100  [Mathematical Formula 1]

In the above formula, absorbance A indicates the absorbance of thepeptide solution measured at 414 nm, absorbance B indicates theabsorbance of PBS measured at 414 nm, and absorbance C indicates theabsorbance of 1% triton X-100 measured at 414 nm.

The hemolytic activity at the concentration of 200 μM, calculated by theformula, is presented in Table 4.

As shown in Table 4, the peptides of the present invention representedrespectively by SEQ. ID. NO: 1, SEQ. ID. NO: 2, and SEQ. ID. NO: 3hardly showed cytotoxicity, while the peptide of the positive controlrepresented by SEQ. ID. NO: 4 clearly displayed significant cytotoxicity(Table 4).

TABLE 4 Hemolysis % SEQ. ID. SEQ. ID. SEQ. ID. SEQ. ID. NO: 1 NO: 2 NO:3 NO: 4 Melittin Ampicillin Oxacillin Hemolytic 0 0 8 71 100 — —activity

Example 4 Analysis of Peptide Aggregation and Structure <4-1>Investigation of Peptide Aggregation by Spectrofluorometer

To investigate whether or not the peptide of the present invention showaggregation in aqueous solution, the present inventors dissolved thepeptide in two aqueous solutions at different concentrations, followedby investigation of peptide aggregation. Tryptophan, one of the aminoacids composing peptide, has the characteristics of emittingfluorescence, by using which aggregation of the peptide can beconfirmed. The peptide was dissolved in water and in PBS at differentconcentrations (1˜18 μM). Changes of fluorescence emission rate wererecorded by using a spectrofluorometer (Perkin-Elmer LS55). Excitationwavelength used for the measurement was 280 nm and emission wavelengthwas 300˜400 nm.

As a result, as shown in FIG. 2A, the maximum fluorescent light emissionwavelength values of those peptides of the present invention representedby SEQ. ID. NO: 3 and NO: 4 did not change over the concentrations (FIG.2A). On the contrary, as shown in FIG. 2B, the maximum fluorescent lightemission wavelength of the peptide represented by SEQ. ID. NO: 4 wasreduced as the concentration of the peptide increased in PBS, while themaximum fluorescent light emission wavelength of the peptide representedby SEQ. ID. NO: 3 did not change in PBS (FIG. 2B). Therefore, it wasconfirmed that the peptide represented by SEQ. ID. NO: 3 did not showaggregation in both aqueous solutions but the peptide represented bySEQ. ID. NO: 4 was aggregated in PBS.

<4-2> Investigation of Aggregation by Circular Dichroism (CD)

Peptide aggregation over different concentrations in PBS was measured bycircular dichroism (CD). Particularly, different concentrations of thepeptide were loaded in CD cuvette, followed by analysis at 190˜250 nm.

As a result, as shown in FIGS. 2C and 2D, the peptide of the presentinvention represented by SEQ. ID. NO: 3 made the consistent graph in PBSregardless of concentrations. In the meantime, the graph representingthe peptide represented by SEQ. ID. NO: 4 showed up and down (FIGS. 2Cand 2D). Therefore, as indicated in the result shown in FIG. 2B, thepeptide represented by SEQ. ID. NO: 3 was not aggregated in PBS, but thepeptide represented by SEQ. ID. NO: 4 was aggregated therein.

Example 5 Confirmation of Active Site of the Peptide in Bacteria

To confirm the active site of the peptide of the present invention in E.coli, the present inventors used confocal laser scanning microscopy. Theantibiotic-tolerant E. coli (E. coli CCARM 1229) and theantibiotic-tolerant Staphylococcus aureus (S. aureus CCARM 3090) werecultured in each optimal medium for them, followed by dilution in PBS atthe density of 2×10⁵ cells/ml. 12.5 μM of the peptide represented bySEQ. ID. NO: 3 labeled with 5-carboxytetramethylrhodamine (TAMRA) wastreated to the E. coli and Staphylococcus aureus, followed by reactionat 37° C. for 10 minutes. Centrifugation was performed at 3000 g for 5minutes and those two bacteria were recovered and washed with PBS, whichprocess was repeated three times. The treated two bacteria were observedunder confocal laser scanning microscope.

As a result, as shown in FIGS. 3A and 3B, the peptide of the presentinvention represented by SEQ. ID. NO: 3 was confirmed to be conjugatedonto the outer wall of the antibiotic-tolerant E. coli and theantibiotic-tolerant Staphylococcus aureus (FIGS. 3A and 3B).

Example 6 Investigation of Effect of the Peptide on E. coli Membrane<6-1> Analysis of Depolarization of Bacteria

To investigate depolarization of bacteria, the antibiotic-tolerant E.coli (E. coli CCARM 1229) and the antibiotic-tolerant Staphylococcusaureus (S. aureus CCARM 3090) were cultured in each optimal medium forthem, and then washed with buffer 1 (20 mM glucose, 5 mM HEPES, pH 7.3)twice. The culture media was diluted with buffer 2 (20 mM glucose, 5 mMHEPES, 0.1 M KCl, pH 7.3) until OD₆₀₀ of each bacterium reached 0.05. 1μM of 3,3′-diethylthiodicarbocyanine iodide (DiSC3-5) was added thereto,followed by reaction until the standard fluorescence was stable. Then,the peptide was treated thereto at different concentrations and thechanges of fluorescence emission rate were recorded. Excitationwavelength used for the measurement was 622 nm and emission wavelengthwas 670 nm.

As a result, as shown in FIGS. 4A and 4B, the peptide represented bySEQ. ID. NO: 1 could not induce depolarization of membrane in bothbacteria, while the peptide represented by SEQ. ID. NO: 2 induceddepolarization approximately 20% at the concentration of 50 μM. In themeantime, the peptides represented by SEQ. ID. NO: 3 and SEQ. ID. NO: 4all induced depolarization approximately 80% at the concentration of 25μM (FIGS. 4A and 4B).

<6-2> Confirmation of Effect on Inner Membrane

To investigate whether or not the peptide of the present inventionaffected the inner membrane, E. coli and S. aureus were cultured in eachoptimal medium for them, and then diluted in PBS at the density of 2×10⁷cells/ml. The cells were reacted with 1 μM SYTOX green (molecular probe)in the darkness for 10 minutes, to which 12.5 μM of the peptide abovewas treated. Thereafter, fluorescence was measured for one hour.Excitation wavelength used for the measurement was 485 nm and emissionwavelength was 520 nm. The value obtained after treating 0.1% tritonX-100 was regarded as 100%, by which the comparative fluorescenceemission rate was measured.

As a result, as shown in FIGS. 4C and 4D, the peptide represented bySEQ. ID. NO: 4 demonstrated the highest fluorescence emission rate atthe time point of 30 min. in E. coli and at the time point of min. inStaphylococcus aureus, while the peptide represented by SEQ. ID. NO: 3demonstrated the highest fluorescence emission rate at the time point of56 min. in E. coli and at the time point of 30 min. in Staphylococcusaureus. The above results indicate that the peptides representedrespectively by SEQ. ID. NO: 3 and SEQ. ID. NO: 4 could affect the innermembrane of E. coli and Staphylococcus aureus (FIGS. 4C and 4D)<

6-3> Confirmation of Time Dependent Apoptosis

To investigate time dependent apoptosis, the antibiotic-tolerant E. coli(E. coli CCARM 1229) and the antibiotic-tolerant Staphylococcus aureus(S. aureus CCARM 3090) were cultured in optimal media respectively. Thebacteria (2×10⁵ cells/ml) were treated with the peptide at theconcentration of MIC and double the MIC, followed by reaction for 0, 1,3, 5, 10, 20, 30, 40, 50, and 60 minutes. A portion thereof was dilutedand smeared on agar media. After culturing the media for 16 hours at 37°C., the number of colonies generated thereon was counted.

As a result, as shown in FIGS. 4E and 4F, the peptide represented bySEQ. ID. NO: 3 demonstrated bactericidal activity after 20 minutes ofculture at the concentration of MIC and after 10 minutes of culture atthe concentration of double the MIC in both bacteria. In the meantime,the peptide represented by SEQ. ID. NO: 4 showed bactericidal activityafter 50 minutes of culture at the concentration of MIC and after 35minutes of culture at the concentration of double the MIC. The aboveresults indicate that the peptide represented by SEQ. ID. NO: 3 works tokill both antibiotic-tolerant bacteria faster than the peptiderepresented by SEQ. ID. NO: 4 does (FIGS. 4E and 4F).

Example 7 Interaction Between the Peptide and Bacteria/Erythrocyte-LikeLiposome <7-1> Investigation of Interaction Between the Peptide andBacteria/Erythrocyte-Like Liposome

To investigate whether or not the peptide of the present inventioninteracted with bacteria and erythrocyte-like liposome, the presentinventors first prepared bacteria and erythrocyte-like liposome. E. colimembrane is mostly composed of L-a-phosphatidylethanolamine (PE) andL-a-phosphatidylglycerol (PG) and erythrocyte (human red blood cell)membrane is composed of egg yolk L-a-phosphatidylcholine (PC) andcholesterol (CH). PE and PG were dissolved at the ratio of 7:3 inchloroform solution. PC and CH were dissolved at the ratio of 10:1 inthe same solution. The prepared solutions were loaded in glass tubes andmoisture was eliminated, followed by freeze-drying. PBS (phosphatebuffered saline, pH 7.4) containing calcein (70 mM) fluorescence samplewas loaded in the glass tube, which was frozen with liquid nitrogen andthen thawed. This freezing and thawing process was repeated 9 times. Toisolate the fluorescence sample that did not flow in liposome, gelfiltration chromatography was performed. The liposome harboringfluorescence sample containing 2.5 μM of lipid was reacted with thepeptide at different concentrations (0.03˜1 μM) for 25 minutes.Fluorescence emitted from the fluorescence sample was measured with aspectrofluorometer. Excitation wavelength used for the measurement was480 nm and emission wavelength was 520 nm. Emission rate measured in thepresence of 0.1% triton X-100 was regarded as 100%, which became thestandard. Based on this standard value, emission rate of each peptidewas calculated according to the below mathematical formula 2 and thencompared.

% fluorescence emission rate=(F−F ₀)/(F _(t) −F ₀)×100  [MathematicalFormula 2]

In the formula, F indicates the absorbance after treated with thepeptide, F₀ indicates the absorbance after treated with buffer, andF_(t) indicates the absorbance after treated with 0.1% triton X-100.Fluorescence emission rate was investigated at different concentrationsof the peptide.

As a result, as shown in FIG. 5A, the peptides represented by SEQ. ID.NO: 3 and SEQ. ID. NO: 4 emitted fluorescence a lot, indicating thatthese peptides interacted with bacteria-like liposome. When comparedbetween these two peptides, as shown in FIG. 5B, the peptide representedby SEQ. ID. NO: 3 emitted fluorescence at most half the level emitted bythe peptide represented by SEQ. ID. NO: 4 at the same concentration(FIGS. 5A and 5B).

<7-2> Investigation of Erythrocyte-Like Liposome Aggregation by thePeptide

To investigate whether or not the peptide of the present invention couldinduce aggregation of erythrocyte-like liposome, PE and PG weredissolved in chloroform at the ratio of 7:3, which was loaded in a glasstube. Moisture was eliminated therefrom, followed by freeze-drying. PBSwas loaded in the glass tube, which was frozen with liquid nitrogen andthen thawed. This freezing and thawing process was repeated 9 times.Then, the solution passed through 0.2 μm filter membrane, and thefiltrate was collected. The peptide was added to 400 μM liposome at theconcentrations of 5, 10, 20, and 40 μM. OD₄₀₅ was measured to examineaggregation.

As a result, as shown in FIG. 5C, the peptide represented by SEQ. ID.NO: 4 induced aggregation of liposome more than the peptide representedby SEQ. ID. NO: 3 did (FIG. 5C).

Example 8 Analysis of Peptide Structure in Bacteria/Erythrocyte-LikeLiposome

To investigate how the structure of the peptide of the present inventioncould be changed in bacteria and erythrocyte-like liposome, the presentinventors first prepared bacteria and erythrocyte-like liposome by thesame manner as described in Example <7-1>. The peptide structure inthose liposome having artificial membrane was analyzed by circulardichroism (CD). 1 mM liposome and 50 μM were mixed, which was loaded inCD cuvette. Absorbance was measured at 190˜250 nm.

As a result, as shown in FIGS. 6A and 6B, the peptides represented bySEQ. ID. NO: 1 and SEQ. ID. NO: 2 did not form any specific structure inthe bacteria-like liposome composed of PE and PG at the ratio of 7:3 norin the erythrocyte-like liposome composed of PC and CH at the ratio of10:1. On the contrary, the peptides represented by SEQ. ID. NO: 3 andSEQ. ID. NO: 4 showed a structural change of folding in both conditions(FIGS. 6A and 6B).

Example 9 Effect of the Peptide on Antibiotic-Tolerant E. coli

To investigate the activity of the peptide of the present invention onthe antibiotic-tolerant E. coli (E. coli CCARM 1229), the presentinventors used scanning electron microscope. The antibiotic-tolerant E.coli was cultured in optimal medium and the cells were diluted in PBS atthe density of 2×10⁷ cells/ml. The peptide represented by SEQ. ID. NO: 3was added thereto at the concentration of 12.5 μM. 60 minutes later,centrifugation was performed at 3000 g for 5 minutes. The precipitatewas washed with PBS and then precipitated again, which was repeatedtwice. The bacteria were fixed with 500 ml of 5% glutaraldehyde in 0.2 Msodium-cacodylate buffer (pH 7.4) at 4° C. for 3 hours and then washedwith 0.1 M sodium-cacodylate buffer. Then, the bacteria were treatedwith 1% osmium tetroxide in 0.1 M sodium-cacodylate buffer, followed byreaction at 4° C. for 1 hour. The bacteria were then washed with thesame buffer containing 5% sucrose twice. Dehydration was performed with20, 40, 60, 80, 95, and 100% ethanol. After drying and coating, thebacteria were observed under scanning electron microscope.

As a result, as shown in FIG. 7 a, the surface of E. coli that was nottreated with the peptide was smooth, but as shown in FIG. 7B, thesurface of E. coli treated with the peptide represented by SEQ. ID. NO:3 was rough and injured, indicating aggregation as well (FIGS. 7A and7B).

The Manufacturing Examples of the composition for the present inventionare described hereinafter.

Manufacturing Example 1 Preparation of Pharmaceutical Formulations <1-1>Preparation of Powders

Peptide of Example 1 2 g Lactose 1 g

Powders were prepared by mixing all the above components, which werefilled in airtight packs according to the conventional method forpreparing powders.

<1-2> Preparation of Tablets

Peptide of Example 1 100 mg Corn starch 100 mg Lactose 100 mg Magnesiumstearate  2 mg

Tablets were prepared by mixing all the above components by theconventional method for preparing tablets.

<1-3> Preparation of Capsules

Peptide of Example 1 100 mg Corn starch 100 mg Lactose 100 mg Magnesiumstearate  2 mg

Capsules were prepared by mixing all the above components, which werefilled in gelatin capsules according to the conventional method forpreparing capsules.

<1-4> Preparation of Pills

Peptide of Example 1   1 g Lactose 1.5 g Glycerin   1 g Xylitol 0.5 g

Pills were prepared by mixing all the above components according to theconventional method for preparing pills. Each pill contained 4 g of themixture.

<1-5> Preparation of Granules

Peptide of Example 1 150 mg Soybean extract  50 mg Glucose 200 mg Starch600 mg

All the above components were mixed, to which 100 mg of 30% ethanol wasadded. The mixture was dried at 60° C. and the prepared granules werefilled in packs.

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended Claims.

What is claimed is:
 1. An antibacterial or fungicidal peptide in which alysine and tryptophan dipeptide (KW) is repeated.
 2. The antibacterialor fungicidal peptide according to claim 1, wherein the lysine andtryptophan dipeptide is repeated 1-10 times.
 3. The antibacterial orfungicidal peptide according to claim 1, wherein the lysine andtryptophan dipeptide is repeated 4 times.
 4. The antibacterial orfungicidal peptide according to claim 1, wherein the peptide is composedof the amino acid sequence represented by SEQ. ID. NO:
 3. 5. Theantibacterial or fungicidal peptide according to claim 1, wherein thepeptide shows antibacterial activity against Gram-positive bacteria. 6.The antibacterial or fungicidal peptide according to claim 5, whereinthe Gram-positive bacteria are selected from the group consisting ofBacillus subtilis, Staphylococcus aureus and Listeria monocytogenes. 7.The antibacterial or fungicidal peptide according to claim 1, whereinthe peptide shows antibacterial activity against Gram-negative bacteria.8. The antibacterial or fungicidal peptide according to claim 7, whereinthe Gram-negative bacteria are selected from the group consisting ofEscherichia coli, Psedomonas aeruginosa and Salmonella typhimurium. 9.The antibacterial or fungicidal peptide according to claim 1, whereinthe peptide has antibacterial activity against antibiotic-tolerantstrains.
 10. The antibacterial or fungicidal peptide according to claim9, wherein the antibiotic-tolerant strains are selected from the groupconsisting of E. coli (E. coli CCARM 1229, 1238), Salmonella (S.typhimurium CCARM 8007, 8009, 8013) and Staphylococcus (S. aureus CCARM3089, 3090, 3108, 3114, 3126).
 11. The antibacterial or fungicidalpeptide according to claim 1, wherein the peptide has fungicidalactivity against pathogenic fungi.
 12. The antibacterial or fungicidalpeptide according to claim 11, wherein the pathogenic fungi are selectedfrom the group consisting of Candida albicans, Candida catenulate,Candida intermedia, Candida rugosa, Candida glabrata and Candidamelibiosica.
 13. The antibacterial or fungicidal peptide according toclaim 1, wherein the peptide has fungicidal activity againstantibiotic-tolerant fungi.
 14. The antibacterial or fungicidal peptideaccording to claim 13, wherein the antibiotic-tolerant fungi are Candidaalbicans (C. albicans CCARM 14001, 14007, 14020).
 15. An antibacterialor fungicidal composition comprising the antibacterial or fungicidalpeptide of claim 1 as an active ingredient.
 16. The antibacterial orfungicidal composition according to claim 15, wherein the composition isselected from the group consisting of an antibacterial or fungicidalpharmaceutical composition, an antibacterial or fungicidal cosmeticcomposition, a non-toxic agricultural pesticide, and a preservative formedicine, cosmetics, food, or feed.
 17. A method for suppressingbacteria or fungi comprising the step of treating a pharmaceuticallyeffective dose of the antibacterial or fungicidal composition of claim15 to the target bacteria or fungi to suppress them.
 18. A method fortreating the pathogenic bacteria or fungi caused disease comprising thestep of administering a pharmaceutically effective dose of theantibacterial or fungicidal composition of claim 15 to a subject havingthe pathogenic bacteria or fungi caused disease.